Cyclosporin A (CsA) is a biologically active product of the fungus Trichoderma polysporum (Calne, R. Y., Immunol. Rev. 46: 113-124, 1979). It is a chemically neutral, extremely hydrophobic cyclic polypeptide composed of 11 amino acids (Petcher, T. J. et al, Helv. Chim. Acta 59:1480, 1976) and has been represented by the following formula A: ##STR1## wherein -MeBmt- represents the N-methyl-(4R)-4-but-2E-en-1-yl-4-methyl-(L)-threonyl residue of the formula B: ##STR2## in which --x--y-- is --CH.dbd.CH--(trans.) (U.S. Pat. No. 4,703,033 to Seebach, P.). The amino acid residues in formula A are referred to by conventional abbreviations, and the same numerical sequence and abbreviations are employed in the specification and claims.
CsA is a potent immunosuppressive agent that is widely used in organ transplantation (White, D. J. G. (ed): Cambridge, England, Elsevier Biomedical, September, 1981; and Cohen, D. J. et al, Ann. Int. Med. 101:667, 1984). The clinical use of CsA, however, is limited by significant CsA toxicity, especially nephrotoxicity, at the recommended therapeutic dosage (Shulman, H. et al, N. Engl. J. Med. 305:1392, 1981; Klintmalm, G. B. G. et al, Lancet, 1:470, 1981; and Kahan, B. D., Transplantation, 40:457, 1985).
CsA in vitro has been found to be extensively metabolized by hepatic cytochrome P-450 microsomal enzymes, and has been found predominantly in its metabolite forms (cyclosporine metabolites) (CM) (Maurer, G. et al, Drug Metab. Dispos. 12:120, 1984; Kahan B. D., Transplant. Proc. 15:446, 1983; and, Maurer, G., Transplant. Proc. 17:19, 1985). CM constitutes well over 70% of cyclosporins found in whole blood, and well over 90% of cyclosporins found in bile of allograft recipients (Cheung, F. Transplant. Proc. 20:602, 1988). The primary metabolites of hepatic CsA metabolism which have been detected are the monohydroxylated derivatives designated OL-17 (in the human), and OL-1 (in the rat), and OL-21 its N-methylated derivative (VenKataraman, R. et al, Transplant. Proc. 20:759, 1988). Other minor CM have been detected; however, their structural identity and purity have hitherto not been established (Cheung, F., Transplant. Proc. 20:602, 1988; and Bowers, L. D. et al, Transplant. Proc. 20:597, 1988).
The role of CM in immunosuppression and toxicity is not clearly documented. CM in vivo have been reported by many investigators to have varying degrees of immunosuppression as compared with the parent CsA. Mauer, G. (Transplant. Proc. 17:19, 1985) Schlitt, N. J. (Transplant. Proc. 19:4248, 1987) and Ryffel, B. (Transplant. Proc. 20:575, 1988) have reported fewer immuno- suppressive effects as compared with the parent CsA. Freed, B. M. (Transplantation 43:123, 1987) have reported comparable effects of OL-17, followed by OL-1 and OL-21, to CsA in the ability to inhibit lymphocyte proliferation in a mixed lymphocyte reaction and IL2 production. The present inventors have reported equal or superior immunosuppressive activity of some CM, as measured both by the ConA proliferative assay and mixed lymphocyte reaction (Dindzans V. and Wong, P. Y., Transplant. Proc. 19:3490, 1987; and Abecassis, M. et al, Can. J. Surg. 31:145, 1988).
Experiments in rats utilizing inducers of the cytochrome P-450 oxidase system have demonstrated that some CM were unlikely to be causative agents of nephrotoxicity (Powell-Jackson, P. R. et al, Transplantation 36:505, 1983, and Goldberg, H. J. Transplantation 47:731, 1989). Ryffel, B. et al (Transplant. Proc. 20:575,1988) have reported that OL-17 was not toxic in vivo in the rat. The present inventors have also shown that some CM are much less toxic to renal and mesangial cells in culture than parent CsA (Cole, et al, Transplant. Proc. 21:943, 1989).
The studies described above are of limited utility in defining the role of individual CM in immunosuppression and toxicity since the composition and purity of many of the CM fractions used in the studies are unknown. As indicated in Bowers, L. D. et al (Transparent. Proc. 20:597, 1988) at page 597, it is imperative that the compounds be isolated and their structural identity and purity be established in order to clearly document the role of CM in the activity and toxicity of CsA.